Structural highlights
Function
[REBH_NOCAE] Involved in the biosynthesis of the indolocarbazole antitumor agent rebeccamycin. Catalyze the chlorination of tryptophan (Trp) at C7 position to yield 7-chlorotryptophan. The reaction between FADH2, Cl-, and O2 generates the powerful oxidant HOCl, which is presumed to carry out the chlorination reaction. The reaction of HOCl with the active site Lys-79 generates a lysine chloramine, which plays a key role in directing regiospecific chlorination of substrate in this important class of biosynthetic enzymes. It is also able to use bromide ions to generate monobrominated Trp.[1] [2] [3]
Evolutionary Conservation
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Publication Abstract from PubMed
The flavin-dependent halogenase RebH catalyzes the formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin. The reaction of FADH2, Cl-, and O2 in the active site generates the powerful oxidant HOCl, which was presumed to carry out the chlorination reaction. Herein, we demonstrate the formation of a long-lived chlorinating intermediate (t1/2 = 63 h at 4 degrees C) when RebH, FADH2, Cl-, and O2 react in the absence of substrate tryptophan. This intermediate remained on the enzyme after removal of FAD and transferred chlorine to tryptophan with kinetically competent rates. The identity of this intermediate is suggested by the X-ray crystal structure of RebH, which revealed an active site Lys79 located in a central position between flavin and tryptophan binding sites and just 4.1 A above C7 of tryptophan. The chlorinating species is proposed to be a Lys-epsilonNH-Cl (lysine chloramine) from reaction of enzyme-generated HOCl with the active site Lys79. This covalent enzyme chloramine likely plays a key role in directing regiospecific chlorination of substrate in this important class of biosynthetic enzymes.
Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases.,Yeh E, Blasiak LC, Koglin A, Drennan CL, Walsh CT Biochemistry. 2007 Feb 6;46(5):1284-92. PMID:17260957[4]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Sanchez C, Butovich IA, Brana AF, Rohr J, Mendez C, Salas JA. The biosynthetic gene cluster for the antitumor rebeccamycin: characterization and generation of indolocarbazole derivatives. Chem Biol. 2002 Apr;9(4):519-31. PMID:11983340
- ↑ Yeh E, Garneau S, Walsh CT. Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis. Proc Natl Acad Sci U S A. 2005 Mar 15;102(11):3960-5. Epub 2005 Mar 2. PMID:15743914 doi:http://dx.doi.org/10.1073/pnas.0500755102
- ↑ Yeh E, Blasiak LC, Koglin A, Drennan CL, Walsh CT. Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases. Biochemistry. 2007 Feb 6;46(5):1284-92. PMID:17260957 doi:10.1021/bi0621213
- ↑ Yeh E, Blasiak LC, Koglin A, Drennan CL, Walsh CT. Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases. Biochemistry. 2007 Feb 6;46(5):1284-92. PMID:17260957 doi:10.1021/bi0621213
